Transcription

and Post-
Transcriptional
Processes
LEARNING GUIDE 2.3
Lesson Objectives

 explain the process of transcription from

the recognition of promoter regions up to
termination; and
 infer the importance of post-
transcriptional processing.
HOOK

 All living organisms have

to have genes
transcribed from their
genomes.
 three stages: initiation,
elongation, and
termination.
Overview of Transcription

 Transcription is the process by

which a segment of DNA directs
the synthesis of an RNA
 DNA does not produce proteins
directly: one strand of DNA must
be copied first into a
complementary RNA sequence.
 RNA polymerase unwinds the DNA
(responsible for polymerization of RNA)
Overview of Transcription

 Unlike DNA replication, the

RNA strand does not remain
hydrogen-bonded to the
DNA template strand in
transcription.
 Also, RNA molecules
synthesized during
transcription are much
shorter than DNA molecules.
Overview of Transcription

 In eukaryotes, the
binding of several
transcription factors that
make the binding of RNA
polymerase possible is
activated by unique
regions in the DNA called
promoters (upstream).
Stages of Transcription
I. Initiation
 requires the DNA double helix to partially unwind
in the region of mRNA synthesis.
 The region of unwinding is called a transcription
bubble.
 The DNA sequence onto which the proteins and
enzymes involved in transcription bind to initiate
the process is called a promoter.
 The structure and function of a prokaryotic
promoter is relatively simple.
 One important sequence in the prokaryotic
promoter is located 10 bases before the
transcription start site (-10) and is commonly
called the TATA box.
Stages of Transcription
I. Initiation
 the RNA polymerase holoenzyme assembles at
the promoter.
 The dissociation of σ allows the core enzyme to
proceed along the DNA template, synthesizing
mRNA by adding RNA nucleotides according to
the base pairing rules, similar to the way a new
DNA molecule is produced during DNA
replication.
Stages of Transcription
I. Initiation
 Only one of the two DNA strands is transcribed.
The transcribed strand of DNA is called
the template strand because it is the template for
mRNA production.
 The mRNA product is complementary to the
template strand and is almost identical to the
other DNA strand, called the non-template
strand, with the exception that RNA contains a
uracil (U) in place of the thymine (T) found in
DNA.
Stages of Transcription

I. Initiation
 Like DNA polymerase, RNA polymerase adds
new nucleotides onto the 3′-OH group of the
previous nucleotide. This means that the
growing mRNA strand is being synthesized in
the 5′ to 3′ direction. Because DNA is anti-
parallel, this means that the RNA polymerase is
moving in the 3′ to 5′ direction down the
template strand
Stages of Transcription

 II. Elongation
 A single gene can be transcribed
simultaneously (i.e. more than one mRNA
molecule are being transcribed at the same
time). DNA is double stranded: sense strand
(coding strand or non-template strand; and
antisense strand (non-coding strand or
template strand).
 RNA nucleotides complementary to the
template strand of the DNA are added to the
3’ end of the growing strand. DNA unwinds 10-
20 bases at a time for pairing with RNA
nucleotides, then the double helix reforms.
Stages of Transcription

 III. Termination
 Transcription stops at the
terminator. In prokaryotes,
termination proceeds through
a termination sequence in the
DNA while in eukaryotes, RNAP
II continues well past the
termination signal.
 Before the
transcription of a
gene is completed, a
protein called Rho
(p) factor binds to
the termination site
and brings about the
termination of
transcription.
Post-Transcriptional Processing in
Eukaryotes
 The RNAP II does not only
transcribes DNA into RNA,
but also bears pre-mRNA-
processing proteins on its
tails, which are then
transferred to the nascent
RNA at the appropriate
time.
 Some regions of the mRNA will not be
translated.
 Some non-coding sequences called
introns are interspersed between
coding (expressed) sequences called
exons.
 The intron loops out as snRNPs (small
nuclear ribonucleoprotein particles;
complexes of snRNAs and proteins)
which bind to signals at the end of
each intron.
 snRNPs join with other proteins to form
a spliceosome. The intron is excised
and the exons are then spliced
together.
 The alteration of mRNA ends includes the
following processes:
 (1) 5’ capping or the addition of a modified G
cap;
 (2) 3’ polyadenylation or the addition of a poly-
A tail; and
 (3) UTRs (untranslated regions) for ribosome
binding signals.
 These modifications share several functions: (1)
to facilitate the export of mRNA from the
nucleus to the cytoplasm; (2) to prevent the
degradation of mRNA by hydrolytic enzymes;
and (3) serve as signals to the rRNA for
attachment to the 5’ end.
KNOT

 Synthesis of RNA is the transcription process, where information is

transferred from a double-stranded DNA molecule to a single-
stranded RNA molecule. This single-stranded RNA is polymerized by
the enzyme RNA polymerase from ribonucleotides on only one of
the two strands of DNA as the template.
 Beyond mRNA processing in eukaryotes, the possible sequence of
events in producing a mature mRNA from a pre-mRNA includes 5’
capping, 3’ maturation (cleavage and polyadenylation), and
splicing. Transcription produces many species of RNA. They vary
from one another in terms of their role in protein synthesis, molecular
size, and tertiary structure.
Translation and
Post-Translational
Processes (Part I)
LEARNING GUIDE 2.4
Lesson Objectives

 enumerate the characteristics of the

genetic code and discuss the implications
of each;
 discuss the different steps involved in
translation; and
 compare and contrast protein synthesis in
prokaryotes and eukaryotes.
The Genetic Code
 The flow of gene-to-protein
information is based on a triplet code
(codon) – a sequence of non-
overlapping, three-nucleotide bases.
 The genetic code is continuous
meaning the start or end of a codon
is not marked by any additional
chemical groups.
 However, it requires instructions for
starting or stopping translation: AUG
(start codon) and UAA, UAG, UGA
(stop codons)
 The Genetic Code
 The genetic code is universal indicating
the same genetic code is used by all
organisms
 It is also redundant, but not ambiguous,
meaning some amino acids are
specified by more than one codon but
no two amino acids are coded by the
same codon.
 These degenerate codons coding for the
same amino acid differ only by the base
in the third nucleotide (wobble effect). In
order for the specified polypeptide to be
formed, codons must be read in the
correct reading frame.
Reading
Genetic
Code
Overview of Translation
 Translation is the synthesis of polypeptide
using the genetic information encoded in
an mRNA molecule. There is a change of
“language” from ribonucleotides to
amino acids.
 Protein synthesis requires multiple
elements: (1) mRNA; (2) tRNA (carrying
amino acids); (3) rRNA (ribosomes); (4)
enzymes and protein factors; and (5)
energy in the form of either ATP or GTP.
 The tRNAs are about 70-80 nucleotides long and
have cloverleaf characteristic structures
resulting from complementary base pairing
between different molecule regions.
 All tRNAs fold into similar compact L shapes,
which are possibly necessary during the
translation process for the tRNAs to fit onto
ribosomes.
 The tRNA interprets the mRNA by acting as an
adaptor molecule that recognizes both the
codon and the encoded amino acid.
 It transfers amino acids to rRNA, hence the
name.
 The anticodon loop located at the other end of
the folded tRNA base pairs with a
complementary codon on the mRNA template.
 The ribosome is a catalytic complex where
protein synthesis takes place.
 A ribosome consists of a large subunit and
a small subunit, each of which is
composed of ribosomal proteins and
rRNAs.
 It facilitates the coupling of tRNA
anticodons with mRNA codons.
 A ribosome has three binding sites for
tRNA: (1) A site (aminoacyl-tRNA binding
site) that holds the tRNA carrying the next
amino acid to be added; (2) P site
(peptidyl-tRNA binding site) that holds the
tRNA carrying the growing polypeptide
chain; and (3) E site that serves as an exit
site for discharged tRNA.
Stages of Translation

 I. Initiation
 The direction of template mRNA for
protein synthesis is 5’ to 3’.
 Stages of Translation
 I. Initiation
 Initiation brings together mRNA, tRNA
with the first amino acid, and the two
ribosomal subunits:
 1. The mRNA binds to the small
ribosomal subunit of rRNA
(ribosomal binding site).

 2. An initiator tRNA anticodon

(UAC) carrying the amino acid
methionine binds to the start
codon (AUG).

 3. The large ribosomal subunit of

rRNA then binds to complete the
translation initiation complex.
Stages of Translation

 II. Elongation
 The direction of the protein synthesized is N-terminal to C-terminal.
During elongation, amino acids are added one by one to the C-
terminus of the growing polypeptide chain. Each addition includes
proteins called elongation factors and takes place in three steps:
 1. Codon recognition. An incoming
aminoacyl tRNA binds to the codon in the A
site.
 2. Peptide bond formation. Peptidyl-
transferase joins the polypeptide from the P
site to the newly arrived amino acid in the A
site.
 3. Translocation. The tRNA in the A site with
the growing polypeptide is translocated to
the P site. The tRNA in the P site moves to the
E site.
Stages of Translation
 III. Termination
 When the stop codon (UAA, UAG, or
UGA) of the mRNA reaches the A site of
the ribosome, the A site recognizes a
protein called release factor instead of
tRNA.
 The release factor hydrolyzes the bond
between tRNA in the P site and the last
amino acid of the polypeptide chain.
 The last tRNA, mRNA and polypeptide
chain are released, the two ribosomal
subunits are separated, and assembly by
other protein factors is dissociated.
Post-Translational Processing

 Attachment of carbohydrates, lipids, phosphate groups

and other functional groups is possible after translation.
 Enzymes may remove one or more amino acids from the
N-terminus of the polypeptide chain. The single
polypeptide chain may also be enzymatically cleaved
into two or more pieces. These two or more separately
synthesized polypeptides can bind together to become
subunits of a quaternary-structure protein.
Post-Translational Processing

 Aided by chaperonins (chaperone proteins), protein folding is

performed simultaneously based on the sequence of amino
acids but it does not specify the final structure of protein.
 On the other hand, ribosomes are interchangeable, meaning
they can be bound (proteins of the endomembrane system and
secretory proteins) or free (cytosolic proteins).
 A signal recognition particle is part of a complex that identifies
the signal peptide and carries the ribosome to the endoplasmic
reticulum through multiprotein translocation.
KNOT

 In protein synthesis, mRNAs code the amino acid sequence, thus representing
transcripts of the structural genes. After transcription, the different mRNAs enter
the cytoplasm where the translation process takes place.
 The tRNAs exist in specific form of each kind of amino acid with unpaired bases
in the anticodon loop that constitute the anticodon. Three binding sites are
established in the large subunit of ribosomes.
 The A site receives a tRNA and binds it in position for complementary base
pairing with the corresponding codon of the attached mRNA. The tRNA at the
P site is released when the peptide bonds are formed, and moves into the E
site and is released into the cytoplasm.